Western Blot Analysis

Cardiac tissues were lysed using radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, and 0.1% sodium dodecyl sulfate (SDS)] containing a protease inhibitor cocktail (Sigma, St. Louis, MO, USA). H9C2 cells were digested by trypsin, and the collected cells were prepared for cell lysis, and proteins were extracted according to the manufacturer’s instruction. Western blotting was performed to detect the levels of various proteins in heart tissue or H9C2 cell lysates quantified by BCA assay. A 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After blocking with 10% skim milk, the membranes were incubated with primary antibodies (TLR4 1:1,000; NF-κB 1:1,000; α-tubulin 1:1,000; β-actin 1:1,000; MMP9 1:1,000; MyD88 1:1,000) at 4°C overnight. After incubation with the appropriate secondary antibodies at room temperature for 1 h, signals were visualized using the enhanced chemiluminescence (ECL) Plus Western blotting detection reagents (Bio-Rad) for 1 min at room temperature. The bands in the membrane were visualized, and densitometric analysis of band intensity was performed using the ChemiDoc Touch Imaging System and Image Lab software (Bio-Rad, Hercules, CA, USA).